Burn wound infection is a major complication leading to patient morbidity and mortality. Topical antimicrobials are cytotoxic to keratinocytes and compromise engraftment of cultured skin substitutes. The goal of this proposal is to generate a therapeutic human skin substitute with antimicrobial activity for use in the treatment of severely burned patients. In the past decade there has been emerging excitement regarding human derived endogenous antimicrobial peptides. One family of peptides, human beta defensins has broad antimicrobial properties against gram positive and gram negative bacteria. The NIKS human keratinocyte cell line is a consistent source of genetically uniform, non-tumorigenic, pathogen free human keratinocytes that will be genetically engineered to express enhanced levels of the broad spectrum defensin family member, hBD-3. Our specific aims are to 1) Develop stable genetically modified NIKS cell clones and evaluate expression levels of hBD-3 and antimicrobial activity in monolayer culture. 2) Produce organotypic cultures using stable hBD-3 clones and characterize based on expression levels, growth properties, ability to form stratified epidermis, and antimicrobial activity. 3) Perform animal grafting for safety and efficacy.